The development of multidrug-resistant strains of Mycobacterium tuberculosis and the emergence of tuberculosis as a major opportunistic infection in HIV- infected people are linked to the worldwide increase in tuberculosis. InhA, the NADH-dependent enoyl-ACP reductase from M. tuberculosis, is the target for the antitubercular drug isoniazid. The enzyme is involved in the biosynthesis of fatty acids and enzyme inhibition interferes with the synthesis of mycolic acid, a critical component of the mycobacterial cell wall. Currently 20% of M. tuberculosis strains are resistant to isoniazid due primarily to mutations in the InhA protein or mutations in the metabolic pathway for isoniazid activation. Consequently, InhA is a bona fide target for the development of novel antitubercular agents. The objective of this proposal is to provide detailed information on the mechanism of InhA with the knowledge that this information will be critical to the design and development of drugs that are effective against both sensitive and drug-resistant strains of M. tuberculosis. Initial efforts are focused on using conjugated substrate analogs to probe the mechanism of InhA. Raman and infrared spectroscopy will provide precise information concerning the geometry and electronic structure of substrates, substrate analogs and cofactors bound to InhA. The vibrational studies will elucidate whether the enzyme promotes catalysis by activating the ground state of the substrate or cofactor to hydride transfer. Site-directed mutagenesis, in concert with the spectroscopic studies, will be used to identify and quantitate the role of specific amino acid residues in substrate activation and transition state stabilization. Structural information on the enzyme- bound ligands will also be provided by NMR spectroscopy using transferred NOE methods and X-ray crystallography. This information will be combined with studies into the molecular basis for isoniazid action to drive the development of novel InhA inhibitors that are effective against both sensitive and drug-resistant strains of M. tuberculosis. Finally, the antimycobacterial activity of InhA inhibitors will be tested directly against cultures of M. tuberculosis.